Antifungal activity of Fe3O4@SiO2/Schiff-base/Cu(II) magnetic nanoparticles against pathogenic Candida species

The antifungal efficacy and cytotoxicity of a novel nano-antifungal agent, the Fe3O4@SiO2/Schiff-base complex of Cu(II) magnetic nanoparticles (MNPs), have been assessed for targeting drug-resistant Candida species. Due to the rising issue of fungal infections, especially candidiasis, and resistance to traditional antifungals, there is an urgent need for new therapeutic strategies. Utilizing Schiff-base ligands known for their broad-spectrum antimicrobial activity, the Fe3O4@SiO2/Schiff-base/Cu(II) MNPs have been synthesized. The Fe3O4@SiO2/Schiff-base/Cu(II) MNPs was characterized by Fourier Transform-Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), Energy-dispersive X-ray (EDX), Vibrating Sample Magnetometer (VSM), and Thermogravimetric analysis (TGA), demonstrating successful synthesis. The antifungal potential was evaluated against six Candida species (C. dubliniensis, C. krusei, C. tropicalis, C. parapsilosis, C. glabrata, and C. albicans) using the broth microdilution method. The results indicated strong antifungal activity in the range of 8–64 μg/mL with the lowest MIC (8 μg/mL) observed against C. parapsilosis. The result showed the MIC of 32 μg/mL against C. albicans as the most common infection source. The antifungal mechanism is likely due to the disruption of the fungal cell wall and membrane, along with increased reactive oxygen species (ROS) generation leading to cell death. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay for cytotoxicity on mouse L929 fibroblastic cells suggested low toxicity and even enhanced cell proliferation at certain concentrations. This study demonstrates the promise of Fe3O4@SiO2/Schiff-base/Cu(II) MNPs as a potent antifungal agent with potential applications in the treatment of life-threatening fungal infections, healthcare-associated infections, and beyond.

The quantitative and qualitative analytical methods were used to characterize and confirm the successful synthesis of the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II).The FT-IR spectra of the materials were recorded on a Tensor II spectrophotometer using the potassium bromide (KBr) pellet at 400-4000 cm −1 (Brucker company).XRD analysis was used to investigate the phase composition and the crystalline structure of MNPs by Bruker AXS D8-Advance X-ray diffractometer with Cu Kα radiation (λ = 1.5418) for 2θ values over the range of 10-80.EDX was utilized to characterize the elements of materials.VSM technique was applied to measure the magnetization of NPs by a BHV-55 VSM using a magnetic field up to 8000 Oe at 300 K. TGA determined the thermal stability of the NPs using a NETZSCH STA 409 PC/PG in the temperature range of 25-750 °C with a heating rate of 10 Antifungal measurement of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) The Minimum Inhibitory Concentrations (MIC) of the six main species of Candida genus fungi were determined using the broth microdilution method.The inocula of tested yeast fungi species from Centraal bureau voor Schimmelcultures (CBS) and American Type Culture Collection (ATCC) including C. dubliniensis (C 8501), C. krusei (A 6258), C. tropicalis (A 750), C. parapsilosis (A 4344), C. glabrata (A 90,030) and C. albicans (C 562) strains, which were prepared from 24-h cultures.Fungal suspensions were adjusted to 0.5 McFarland standard turbidity which is a stock suspension of 1-5 × 10 6 cells/mL.For determination of antifungal activities, serial dilutions of the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) (0.5 to 256 μg/mL) were prepared in Roswell Park Memorial Institute (RPMI-1640) medium, in 96-well microtiter plates.Then 100 μL of the working inoculums of tested yeast fungi were added to the microtiter plates and incubated in a humid atmosphere at 32 °C for 24-48 h.The culture medium alone and culture medium with inoculums (yeast) were adjusted as blank and growth controls, respectively.It is noteworthy that each experiment was carried out in triplicate.After incubation time, the existence of growth in 96-well microtiter plates was compared with the growth control.The lowest concentration of the mentioned treatments, without visible growth, was considered MIC.Additionally, 10 μL medium from no visible growth of yeast fungi wells on Sabouraud Dextrose Agar (SDA) was applied to specify Minimum Fungicidal Concentration (MFC).The MFC values were assessed as the lowest concentrations producing no more than 4 colonies (demonstrating 99.9% mortality of the fungi in the initial inoculums) 39,40 .

The MTT assay
The cytotoxicity of the produced Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) NPs was measured quantitatively using the MTT test 41 .At a density of 5 × 10 3 cells per well in a 96-well cell culture plate (5000 cells/96 well), the mouse L929 murine fibroblastic cell line was grown at 37 ˚C with CO 2 in a humidified incubator containing DMEM/ F12 (1:1 mixture of Dulbecco's Modified Essential Medium (DMEM) and Ham's F-12 Medium) culture media supplemented with 10% (v/v) fetal bovine serum (FBS), 100 unit/ml of penicillin and 100 µg/ml of streptomycin.16, 32, and 64 μg/mL of sterilized Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs were added to each row (8 replicates).No treatment was added to one row and it was considered as the control group.At each time point (1, 3, and 5 days after cells seeding), the culture medium was removed and 0.2 ml of MTT (0.5 mg/mL) was added to each well, and the cells were incubated at 37 ˚C for 4 h in an incubator in dark condition.Then, the solution was removed and 0.1 mL DMSO was added to each well to dissolve the formed formazan crystals.The absorption values (OD) of the samples were measured at a wavelength of 570 nm utilizing a microplate reader of Biotech Instruments.

Results and discussion
Preparation of the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs As described in the experimental section, the preparation of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs is briefly shown in Fig. 1.First, the coating of the Fe 3 O 4 core NPs being formed by the combination of Fe (II) and Fe (III) chloride salts was done by using the silica source of TEOS under PH control to afford Fe 3 O 4 @SiO 2 core-shell NPs.Second, the Schiff base complex of Cu(II) was made through the reaction between salicylaldehyde and APTES followed by adding Cu(OAc) 2 .4H 2 O. Finally, the Schiff base complex of Cu(II) was immobilized on the surface of Fe 3 O 4 @SiO 2 NPs under reflux conditions to obtain the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs (Fig. 1).SiO 2 /Schiff-base/Cu(II) appeared at distinctive vibrational bands of 520 and 580 cm −1 , respectively (Fig. 2e  and a).Also, the Fe 3 O 4 @SiO 2 and Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) showed the characteristic band of Si-O-Si at around 1200 cm −1 (Fig. 2b and e).The C = N bond in the Schiff-base ligand represented the stretching band at 1631 cm −1 (Fig. 2c), which appeared at the lower frequency of 1618 cm −1 in both the Cu(II)/Schiff-base complex (Fig. 2d) and Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) (Fig. 2e) due to the anchoring of Cu metal in the complex.Based on this observation, the synthesis of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) has been verified by the FT-IR evidence.3g) 32 , which appears at lower angles due to the binding of Cu(II)-Schiff base complex to the Fe 3 O 4 @ SiO 2 (Fig. 3h).So, the XRD outcome supports the efficient immobilization of the Cu(II)-Schiff base complex on the Fe 3 O 4 @SiO 2 MNPs without any alteration in the structure of Fe 3 O 4 MNPs.

SEM, TEM, DLS results
TEM, SEM, and DLS results of Fe 3 O 4 , Fe 3 O 4 @SiO 2 , and Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs are depicted in Fig. 3.The TEM image of Fe 3 O 4 displays the harmonic dark spheres with an estimated particle size of 10-15 nm (Fig. 3a).The grey silica layer on the surface of Fe 3 O 4 NPs results in a TEM image with maintaining the spherical pattern, which makes the Fe 3 O 4 @SiO 2 MNPs of 20-25 nm in size (Fig. 3b).The immobilization of Cu(II)/ Schiff-base complex on the Fe 3 O 4 @SiO 2 NPs did not affect the spherical structure pattern and an average size of 30-38 nm was obtained for the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs (Fig. 3c).The uniform spheres and morphological patterns are shown in the NPs (Fig. 3d-f), which confirms the effective surface modification of the Fe 3 O 4 MNPs with silica layer and the Fe 3 O 4 @SiO 2 with Cu metal complex afterward 34 .The mean particle size distribution of Fe 3 O 4 , Fe 3 O 4 @SiO 2 , and Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs was measured by the DLS analysis, which shows the average size of 12, 20, and 32 nm, respectively (Fig. 3g-i).

EDX spectrum
The EDX analysis revealed the elemental composition in the (a) Fe

VSM and TGA analyses
The superparamagnetic behavior of Fe 3 O 4 , Fe 3 O 4 @SiO 2 , and Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs was studied by VSM analysis at room temperature.A magnetic field up to 8000 Oe at 300 K was applied to investigate the super-paramagnetization (Fig. 6a-c).The magnetization feature of the NPs is confirmed by observing no hysteresis phenomenon in the magnetization curves.The saturation magnetization (Ms) values of Fe 3 O 4 , Fe 3 O 4 @ SiO 2 , and Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs were found to be 68 (Fig. 6a), 45 (Fig. 6b), 34 (Fig. 6c) emu/g, respectively.These slight decreases in the Ms values of NPs stem from the functionalization of Fe 3 O 4 with SiO 2 core-shell and Cu(II)-Schiff base complex, subsequently.However, it does not have an influential effect on the magnetization identity of NPs, especially Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) 33 .This evidence is finally confirmed by evaluating the magnetization of NPs with an external magnetic field (Fig. 6d).The Fe 3 O 4 @SiO 2 /Schiff-base/ Cu(II) would be separated simply and rapidly, which is an essential factor in retrieving the MNPs.
The thermal stability of Fe 3 O 4 , Fe 3 O 4 @SiO 2 , and Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs was analyzed by TGA in the temperature range from 25 to 750 °C (Fig. 6e-g).The thermograms show the fraction of volatile components by increasing the temperature.Negligible mass loss is observed in the Fe 3 O 4 and Fe 3 O 4 @SiO 2 slopes until higher temperatures reveal the thermally stable structure of the NPs (Fig. 6e,f).The removal of adsorbed water, intact organic solvents, and hydroxyl groups in the range of 100 °C and 500 °C contributes to this slight decrease 32,37 .Also, a decrease in the slope appears in the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs until 300-350 °C, after which the mass loss is attributed to the decomposition of organic compounds (Fig. 6g).This observation verifies both the successful immobilization of Cu(II)/Schiff-base complex on the Fe 3 O 4 @SiO 2 MNPs and the excellent thermal resistance of the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs at high temperatures.moderate to good stability of the Cu MNPs and the negative charge on the surface of Cu MNPs.This major factor contributed to the stability of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs in suspension, and successful initial adsorption onto the cell membrane of fungi infections.

Antifungal activity of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II)
Table 1 represents the MIC and MFC values of the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) towards the studied fungal species.In this study, the antifungal activity of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) was investigated against 6 Candida species.The Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) inhibited the growth of all of the examined yeast strains at concentrations of 8-64 μg/mL.Throughout the study, C. parapsilosis had the lowest MIC and MFC, which were 8 and 16 µg/mL, respectively while C. krusei exhibited the highest MIC and MFC (64, 256 µg/mL).As a result, C. parapsilosis was the most susceptible to Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II).In simpler words, the Fe 3 O 4 @SiO 2 / Schiff-base complex of Cu(II) is more effective in inhibiting or killing C. parapsilosis.
This efficacy might stem from the small particle size of the Cu NPs, leading to a gradual release of the therapeutic agent.Although fluconazole as a standard drug, shows higher effectiveness in some instances, as indicated by MIC values, the slow-release characteristic of Fe 3 O 4 @SiO 2 /Schiff-base complex of Cu(II), along with a reduced propensity to cause drug resistance, could render it a more viable option for long-term treatment.This aspect is especially crucial for treating persistent fungal infections and in scenarios where resistance to fluconazole is a concern, potentially enhancing therapeutic effectiveness.
As depicted in Fig. C. albicans is a common cause of candidemia, and its resistance to some antifungal drugs such as fluconazole can limit treatment options.It can cause a wide range of clinical manifestations and easily spread through the hands of healthcare workers, leading to nosocomial infections 42 .To address this problem, the obtained result reveals the potential of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) can be an efficient candidate based on the obtained results.Therefore, regarding C. albicans as the most frequent fungus causing a range of infections from mild skin and mucosal infections to life-threatening invasive infections 43 , the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) showed satisfactory antifungal activity.
Significantly, the creation of the Fe 3 O 4 @SiO 2 @Schiff-base/Cu (II) MNPs has led to an innovative approach in controlled drug release and precise drug targeting for localized fungal infections, including fungal endocarditis.This is achieved through the application of an alternating current magnetic field.The magnetic characteristics of Fe 3 O 4 @SiO 2 @Schiff-base/Cu (II) might enhance the accurate delivery of the therapeutic agent to the affected tissues.This can be accomplished using an external magnetic field, either on its own or in conjunction with other medicinal drugs 44 .

Antifungal mechanism of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs
The fungal cell constitutes a wide range of substructures, in which the cell membrane, cell wall, ribosomes, mitochondria, storage vacuoles, Golgi bodies, and DNA are vital organelles (Fig. 9a) 45 .The hypothetical antifungal mechanism of Cu-NPs is depicted in Fig. 9b.Although the antifungal activities of different Cu-NPs have been studied, the precise mechanism of action for NPs is not yet recognized.Based on previous reports, the cell wall of fungi can be destroyed through protein denaturation by confronting Cu-NPs, which occurs through interaction     between the surface of fungi covered with carboxyl, amino, and sulfhydryl groups in the peptidoglycan layer and Cu-NPs 46 .Having been penetrated to the cell wall by endocytosis, the Cu-NPs damage the cell membrane leading to decreasing the electrochemical potential and destroying the integrity, subsequently 46 .Releasing the Cu ions in fungi cells is followed by the generation of ROS.The ROS paves the way for DNA fragmentation, enzyme activity inhibition, ribosome and protein denaturation, mitochondria damage, and affecting other components, which ends in cell death, consequently 47 .
It is noteworthy that the antifungal activity of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) against Candida species can be attributed to several factors.Firstly, the presence of Cu(II) ions in the composite material can disrupt the cell membrane of Candida species, leading to cell death.Secondly, the Schiff base ligand in the composite material can be able to block a specific metabolic pathway that is essential for the microorganism's survival and enhance its antifungal activity, subsequently.Additionally, the Fe 3 O 4 @SiO 2 core-shell structure provides a large surface area for interaction with Candida cells, increasing the effectiveness of the antifungal activity of this product 48 .Besides, the magnetic properties of Fe 3 O 4 allow to use of a magnetic field by which the drug can be directed to the site of infection, and a lower dose of the drug sets the stage for improving the therapeutic efficacy and reducing toxicity 39 .

Cell viability and proliferation assessment
An MTT assay was carried out to further explore the L929 cell proliferation with Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs on days 1, 3, and 5.As shown in Fig. 10a, a steady upward trend can be observed for 32-and 64-μg/mL samples over time.The cell viability in the 16-μg/mL sample increased on the two first days despite a minor decrease observed on the 5th day.The only significant difference between the groups was observed on the first day when the OD value of 64-μg/mL sample was less than the control group.Based on these findings, the results of the cell proliferation assay revealed that the incorporation of Cu within NPs not only had no cytotoxic effect on cells but triggered the growth of cells containing the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs in almost all days as well as the control group.Figure 10b indicates a schematic view of the MTT test and cell growth observation over a specific time.

Conclusion
Herein, we successfully prepared the Schiff-base ligand of Cu(II) supported on the Fe 3 O 4 @SiO 2 MNPs using the co-precipitation method and simple immobilization.The characterization results confirmed the specific absorption peaks, the maintenance of the spinel crystalline pattern, magnetic behavior, thermal stability, nanosized and morphological structure of the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs by FT-IR, XRD, VSM, TGA, TEM, SEM and DLS techniques.The striking feature of the study aimed at the antifungal ability of Fe 3 O 4 @ SiO 2 /Schiff-base/Cu(II) MNPs against C. dubliniensis, C. krusei, C. tropicalis, C. parapsilosis, C. glabrata and C. albicans strains.The microdilution method using 0.5-256 μg/mL dilutions of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs as antifungal agent revealed the lowest inhibition and fungicidal concentrations at 8 and 16 µg/ml for C. parapsilosis.The opposite result was recorded for C. krusei with the highest MIC and MFC (64, 256 µg/mL).The growth of C. Albicans as the leading cause of fatal infections was inhibited at the concentration of 32 µg/ml
3 O 4 , (b) Fe 3 O 4 @SiO 2 and (c) Fe 3 O 4 @SiO 2 / Schiff-base/Cu(II) (Fig. 4).The characteristic elements of Fe and O in Fe 3 O 4 are depicted in Fig. 4a.The presence of a large amount of Si in the elemental composition spectrum of Fe 3 O 4 @SiO 2 shows the successful coating of Fe 3 O 4 NPs by the silica layer.(Fig. 4b) 32 .As depicted in Fig. 4c, the elemental peaks of Fe, O, C, N, Si, and Cu in the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) indicate the efficient functionalization of Fe 3 O 4 @SiO 2 with the

Fe 3 O 4 @
SiO 2 /Schiff-base/Cu(II) According to Fig. 7, the zeta potential magnitude of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs obtained −31.8 mV, which indicates the stability of colloidal dispersions and the surface charge.The value of -31.8 Mv indicates the
8, C. parapsilosis placed first in treating with the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs with the lowest amount of 8 μg/mL.C. glabrata ranked the second position with the amount of 16 μg/mL of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs.C. albicans, C. tropicalis and C. dubliniensis held the subsequent positions with the same amounts of antifungal agent (32 μg/mL).C. krusei occupied the last position, for which 64 μg/mL of the Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) MNPs for inhibition was used.

Figure 10 .
Figure 10.Cell viability (MTT assay): (a) the proliferation of control and different concentrations of samples during the incubation times of the first, third, and fifth day, (b) representation of cell plates with different concentrations.

Table 1 .
Antifungal effects of Fe 3 O 4 @SiO 2 /Schiff-base/Cu(II) on the fungi strains based on broth microdilution method.